With the increase in the smoking population and the aggravation of air pollution, lung cancer cases have rapidly increased in South Korea. A Korean statistical report has it that lung cancer cases rank first after stomach cancer in men and come after breast cancer, uterine cancer, stomach cancer and colorectal cancer in women, and is associated with the highest mortality in both men and women. The frequency of incidence of lung cancer and the mortality rate from lung cancer in Korea are expected to increase for a significant period of time in the future in light of the current trend of the smoking rate.
There are two major cell types of lung cancer, small cell lung carcinoma and non-small cell lung carcinoma. Non-small cell lung carcinoma accounts for about 80% of lung cancer cases and is further divided into the three main subtypes of adenocarcinoma, squamous cell carcinoma and large cell carcinoma.
As the cells grow, the lung cancer causes various symptoms including vascular invasion into adjacent tissues, airway blockage, metastasis to lymph nodes, etc. Approximately 10˜15% of lung cancer cases without any symptoms are diagnosed during a regular checkup. In most cases, lung cancer, when diagnosed, has already progressed to stage III or beyond, so that they are, for the most part, difficult to cure. Therefore, early diagnosis of lung cancer is presenting itself as an urgent problem to be solved so that the mortality from lung cancer can be reduced.
Various methods are used in combination to diagnose lung cancer. So far, a number of lung cancer screening tests have been employed, including examination of tumor size, metastasis to lymph nodes, immunohistochemistry of biopsy samples of tumerified lung tissue or lymph nodes, chest X-ray screening, chest computerized tomography, and bronchoscopy. Lung cancer can be shown in chest computerized tomography only if the size of the tumor is 0.1 cm or larger. However, lung cancer with this size has probably already metastasized to other tissues. In bronchoscopy, the inside of the lung can be directly observed with an endoscope, but there is the problem of spatial limitation that makes it difficult to observe tumors in deep places.
To supplement such lung cancer screening methods, attempts have been made to use the levels of CBC (complete blood count), serum electrolytes (including calcium), alkaline phosphatase, albumin, AST (aspartate aminotransferase), ALT (alanine transaminase), total bilirubin or creatinine, to diagnose breast cancer. Although their potential values as diagnosis or prognosis factors, the application of conventional tumor biomarkers has shown limit in specificity and sensitivity, and there are no officially recommended breast cancer markers.
The most common cell type of colorectal cancer is adenocarcinoma from the view of pathology. Colorectal cancer is broadly divided into colon cancer and rectal cancer depending on the site of the lesion. As high as about 50% of colorectal cancer cases occur in the lower tract of the large intestine, that is, the rectum. Recent research data indicate that the incidence of colorectal cancer and the mortality from colorectal cancer have significantly increased in Korea, with the westernization of dietary habits. Risk factors for colorectal cancer, although remaining unclear, include heredity, dietary habits associated with the intake of high-fat and low-fiber diet, and inflammatory bowel disease.
Colorectal cancer may attack persons of all ages, but the risk of developing colorectal cancer increases with age and most cases occur in patients in their 60s and 70s. Colon cancer occurs at a relatively higher rate in women than in men, but in the case of rectal cancer, the reverse is true.
To treat colorectal cancer, colectomy is predominantly used in combination with chemotherapy and radiotherapy. In spite of advances in surgical therapy, chemotherapy and radiotherapy, the mortality from colorectal cancer is high because colorectal cancer is caught at too late of a stage for it to be surgically operated on in many cases because the cancer progresses without any particular symptoms. Colorectal cancer patients have an average 5-year survival of approximately 90% or higher for stage I, approximately 70% or higher for stage II, and approximately 50% for stage III whereas the average 5-year survival decreases to approximately 5% in the case of stage IV (2004 Information on Cancer, published by the National Cancer Center, Korea). When colorectal cancer is detected in the earlier stages, it is more likely to be curable, resulting in a greater increase in survival rate.
Screening for colorectal cancer is recommended in individuals who are at high risk. There are several different tests available for this purpose, including rectal digital examination, fecal occult blood test and colonography. The earliest detection procedures presently available for, colorectal cancer involve using tests for fecal blood or endoscopic procedures. However, the size of the tumor must be significant before fecal blood is detected. The sensitivity of the guaiac-based fecal occult blood tests is as low as approximately 26%, which means the 74% of patients with malignant lesions will remain undetected. The visualization of precancerous and cancerous lesions represents the best approach to early detection, but colonoscopy is invasive and accompanied by significant costs, risks, and complications.
Such conventional methods suffer from the disadvantages of having poor accuracy in diagnosis, being unable to diagnose colorectal cancer in its pre-stages and being unable to predict the onset of colorectal cancer, and inconveniencing patients. As a supplement for such conventional colorectum screening methods, attempts have been made to use blood tumor marker levels to diagnose colorectal cancer.
At present, primarily diagnostic blood tests based on the detection of carcinoembryonic antigen (CEA), a tumor-associated glycoprotein, are available to assist with diagnosis in the field of colorectal cancer. Carcinoembryonic antigen is increased in 95% of tissue samples obtained from patients with colorectal, gastric, and pancreatic cancers and in the majority of breast, lung, and head and neck carcinomas. Elevated carcinoembryonic antigen levels have also been reported in patients with non-malignant disease, and many patients with colorectal cancer have normal CEA levels in the serum, especially during the early stage of the disease. The utility of carcinoembryonic antigen as measured from serum or plasma in detecting recurrences is reportedly controversial and has yet to be widely applied.
Therefore, there is long lasting unmet need for a biomarker that allows lung cancer and colorectal cancer to be accurately diagnosed in their early stages and which improves the chance of survival for the patients.
Glutaredoxins (Glrx) are small redox enzymes of approximately 100 amino acid residues that use reduced glutathione as a cofactor in reducing various substrates by a dithiol-monothiol mechanism. There are two main groups of glutaredoxins divided into dithiol glutaredoxins with the characteristic active site consensus sequence Cys-Pro-Tyr-Cys and monothiol glutaredoxins with a Cys-Gly-Phe-Ser active site consensus sequence which lacks one C-terminal active site thiol. Monothiol glutaredoxins can be further categorized into single domain monothiol glutaredoxins and multi-domain glutaredoxins. The single domain monothiol glutaredoxins consist of only one glutaredoxin domain while the multi-domain monothiol glutaredoxins contain an N-terminal thioredoxin-like domain and one to three C-terminal monothiol glutaredoxin domains. The multi-domain monothiol glutaredoxins are restricted to eukaryotic cells while dithiol glutaredoxins and single domain monothiol glutaredoxins are common to all living organisms.
Glutaredoxins are divided into glutaredoxin 1, glutaredoxin 2 glutaredoxin 3 and glutaredoxin 5 . Human glutaredoxin 1 and glutaredoxin 2 are respectively cytosolic and mitochondrial isoforms. Human glutaredoxin 3 is a multi-domain monothiol glutaredoxin and human glutaredoxin 5 is a single domain monothiol glutaredoxin located in the mitochondria.
Cellular redox states are associated with major cellular processes including differentiation, transformation, and apoptosis. Human glutaredoxin 1 binds to mitogen-activated protein kinase kinase kinase 5 (MAP3K5 also known as apoptosis signal-regulating kinase 1) dependent on its redox status. In this complex, the kinase activity of MAP3K5 is suppressed. Oxidation of glutaredoxins leads to the dissociation of the complex and the activation of MAP3K5. Hence, glutaredoxin 1 may regulate MAP3K5's kinase activity in response to the glutathione redox state. In addition, glutaredoxin 1 protects cells from hydrogen peroxide-induced apoptosis by regulating the redox state of protein kinase B.
Glutaredoxin 2 protects HeLa cells from oxidative stress-induced apoptosis.
Glutaredoxin3 was identified as a protein kinase C theta (PRKCQ)-interacting protein that inhibits the activation of mitogen-activated protein kinase 8 (MAPK8 also known as c-Jun N-terminal kinase) stimulated with PMA (phorbol myristate acetate), ultraviolet radiation, or transcription factors jun oncogene (JUN, also known as AP-1) and NF-κB (nuclear factor kappa-B) in Jurkat T cells. In a subsequent study, the treatment of Jurkat T cells with hydrogen peroxide induced the tyrosine phosphorylation of glutaredoxin 3 in a dose-dependent manner that was also dependent on LCK (lymphocytespecific protein tyrosine kinase). MAP3K5 activates the MAPK8 and MAPK14 pathways and is required for tumor necrosis factor α-induced apoptosis. These observations suggest that glutaredoxin 3 inhibits apoptosis via its role in cell activation-associated signaling pathways or in the cellular response to stress signals.
Thus, these data indicate the involvement of glutaredoxins in the process of oncogenesis. However, only a few studies report the association of glutaredoxin 1 with cancer. Overexpression of glutaredoxin 1 was found to increase the resistance of MCF7 breast cancer cells to doxorubicin, a widely used anti-cancer agent. An immunohistochemical analysis by Nakamura et al. revealed increased expression of glutaredoxin 1 in cancerous pancreatic tissue.
As mentioned above, there are reports on the association of glutaredoxin 1 with carcinogenesis, but the association of glutaredoxin 3 with cancer has been little studied. In fact, almost no studies have been done on the use of glutaredoxin 3 as a diagnostic biomarker for cancer, especially lung cancer and colorectal cancer. In this invention, glutaredoxin 3 is used as a diagnostic biomarker for lung cancer and colorectal cancer that allows the accurate early diagnosis and prognosis of lung cancer and colorectal cancer.